Chromatographic separation of target molecules is of great commercial interest in the chemical and biotechnological fields, such as the large-scale production of novel biological drugs and diagnostic reagents. Furthermore, the purification of proteins has recently become of great significance due to advances in the field of proteomics, wherein the function of proteins expressed by the human genome is studied.
In general, proteins are produced in cell culture, where they are either located intracellularly or secreted into the surrounding culture media. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, growth factors, etc. Separation and purification of a desired protein from the complex mixture of nutrients and cellular by-products, to a level sufficient for therapeutic usage, poses a formidable challenge.
Porous polysulphone and cellulosic membranes are widely used for filtering and separating chemical and biological mixtures (cf. EP 0483143). These membranes include ultra- and microfiltration membranes, in which the filtration process is based on a hydrostatic pressure differential. Ultra-filtration membranes are characterised by pore sizes which enable them to retain macromolecules having a molecular weight ranging between 500 and 1,000,000 daltons. Microfiltration membranes exhibit permselective pores ranging in diameter between 0.01 and 10 μm.
Cellulosic hydrate and ester membranes are well known in the membrane filtration art and present a unique combination of advantageous characteristics, including hydrophilicity, which permits wettability without the use of surfactants. Such membranes also exhibit minimal protein adsorption, high resistance to heat and a high degree of flexibility.
However, despite their widespread usage, cellulosic membranes suffer a number of disadvantages, including susceptibility to attack by strong acids and bases, and by cellulase enzymes. Sensitivity to bases is characterised initially by shrinkage and swelling, ultimately leading to decomposition of the membrane. High temperatures promote chemical disintegration and shrinkage while low temperatures, especially in connection with substantial concentrations of alkali, promote swelling and bursting. The pore structure of the membrane can easily be destroyed resulting in a dramatic decrease in the flow rate through the membrane. The alkali sensitivity of cellulose membranes is a marked disadvantage when, for example, strongly alkaline cleaning media are required to clean the membrane to restore its filtration capacity.
Cellulases are encountered in the brewing industry, and also develop spontaneously from microorganisms that grow on cellulose membranes during prolonged storage in a non-sterile environment. Cellulases attack the membranes by decomposing the cellulosic polysaccharides therein into smaller chemical fragments such as glucose. When cellulose hydrate membranes decompose, some of the byproducts of the decomposition lead to the formation of so-called “pseudopyrogens” or fever-producing substances which mitigates against the use of cellulose hydrate membranes in the filtration of pharmaceutical products.
From the experience of the textile industry, it has long been known that better characteristics may be imparted to cellulosic fibers by cross-linking (cf. Kirk-Othmer's Encyclopedia of Chemical Technology, Vol. 22, pp 770-790 (3rd Ed. 1983)). Such cross-linking is particularly desirable in order to improve the physical strength and chemical resistivity of the cellulosic membranes. Furthermore, where chemical derivitization of the membranes is desirable, for example in order to couple protein binding ligands to the hydroxyl groups of the cellulose polymers, base sensitivity is particularly important.
A process for cross-linking regenerated cellulose hydrate membranes, for use in the separation of ketone dewaxing solvents from dewaxed oil, is disclosed in EP 0145127, the process comprising contacting cellulose hydrate membranes with a solution of a cross-linking agent. However, the cross-linked membrane products exhibited considerable degradation in their hydrophilic properties as compared to the original membrane. Moreover, with increased cross-linking, the flux of such membranes dramatically decreases by about 80% compared to the flux of non-cross-linked cellulose hydrate membranes. Furthermore, cross-linking with the bifunctional reagents, because of their low water-solubility, required the use of organic or aprotic solvents, which makes the process technically difficult and expensive.
EP 0214346 describes a process for cross-linking cellulose acetate membranes, to enhance their resistance to organic liquids, for use in the separation of polar solvents such as ketone dewaxing solvents present in dewaxed oil. Cross-linking is achieved by use of bifunctional reagents which are reactive with the hydroxyl groups present in the structure of the cellulose acetate membrane. It should be noted that the bifunctional reagents react directly with the free hydroxyl groups present in the cellulose acetate membrane, there being no disclosure of any removal of the acetate groups by base hydrolysis in the document.
U.S. Pat. No. 5,739,316 teaches a process for cross-linking cellulose hydrate membranes with a water-soluble diepoxide (such as 5-ethyl-1,3-diglycidyl-5-methylhydantion) in the presence of a base. The alkaline medium acts as a catalyst for the reaction of the diepoxide with the cellulose and also in deactivating the adverse effect water has on the cellulose. Applications cited for the membranes include use in the separation of aqueous/oil emulsions and the separation of proteins from biotechnically produced aqueous media and beverages.
A process by which cross-linked cellulose hydrate membranes are produced is disclosed in US 2004/0206694. A regenerated cellulose hydrate membrane is treated with epichlorohydrin under basic and reducing conditions to yield an epoxidised cross-linked product. This product may be further treated with a nucleophilic amine reagent (e.g. dimethylethylenediamine) to provide a positively charged cross-linked cellulose membrane. Alternatively, a negatively charged membrane may be obtained by reaction of the epoxidised cross-linked product with sodium chloroacetate under basic conditions.
A one step process for producing positively or negatively charged membranes is also described in which glycidyl reagents having epoxide groups and groups capable of possessing charge (e.g. glycidyl quaternary compound or glycidyl acid) can be reacted directly with hydroxyl polymers under basic conditions.